The long-term objective is to clarify the nutritional requirements of the blastocyst period in mammalian development, so that the successful maintenance of normal blastocysts may be accomplished in vitro. The specific aims focus on an aspect of blastocyst metabolism that has received little attention in the past: its high level of oxygen consumption and the potential damage from reactive metabolites of oxygen. The work will utilize blastocysts of the rabbit, since their rapid expansion and increase in cellular mass during this period of development may make them highly susceptible targets for superoxide anions, peroxides, and hydroxyl radicals. Cyanide-resistant reduction of nitroblue tetrazolium is readily demonstrable in rabbit blastocysts, as it is in activated phagocytes which are known to be producing lethal amounts of these reactive groups. The premise is that in vivo the blastocyst satisfactorily "scavenges" these radicals and that the uterine milieu plays a role in this process. The work will require exposing rabbit morulae to various culture media during the initial 48 hours of expansion. Physiological scavengers such as reduced glutathione and alpha-tocopherol will be tested for their ability to promote normal expansion of the blastocyst, mitotic rates in the trophoblast, early differentiation of the embryoblast, and implantation and continued normal development in embryos reimplanted in a pseudopregnant uterus. Another aspect of blastocyst metabolism to be investigated is the requirement for iron and the role of transferrin uptake in normal preimplantation development. Previous work has shown active replication and transcription of mitochondrial DNA in the rabbit blastocyst, and iron is required for assembly of the electron transport chain. Ultrastructural evidence suggests that transferrin receptors appear on rabbit trophoblast early in the blastocyst period, and purified transferrin is a satisfactory substitute for whole serum in blastocyst culture. Again, morulae will be maintained for 48 hours in defined media containing various levels of transferrin and assayed as above for normality following the culture period. Hormonally-induced superovulation will be employed as necessary to provide sufficient numbers of experimental and control embryos for statistical evaluation of the culture conditions tested.